Anti-tumor substance of immunoglobulin and melphalan and process for producing the same

ABSTRACT

Disclosed herein are an anti-tumor substance obtained by bonding an anti-tumor agent to human immunoglobulin, and a process for producing the same.

This application is a continuation of application Ser. No. 595,037,filed Mar. 29, 1984, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to an anti-tumour substance obtained bybonding an anti-tumour agent to human immunoglobulin and a process forpreparing the same.

Recently, with the development of immunochemistry, a large number oftumour-related antigen have been discovered and the tumour-specificantibody which selectively bonds to these tumour-related antigens hasbeen developed. Further, trials are now under way wherein an anti-tumouragent is bonded to the tumour-specific antibody and the thus formedsubstance is administered to the patient suffering from the tumour fortransferring the substance collectively to the portion of the tumour. Inthis trial, the tumour-specific antibody is prepared by a technique ofimmunizing a rabbit, horse and sheep with the tumour cells or thetumour-related antigen and is obtained from the serum of the thusimmunized animal as an immunoglobulin fraction. In more recent years,after immunizing a mouse with the tumour cells or the tumour-relatedantigen, the antigen-producing cells are taken out from the mouse andthe thus collected cells are subjected to cell fusion with themouse-myleloma cells such as NS-1, thereby obtaining the monochronalcells producing anti-tumour antibody, and the anti-tumour antibody istaken out from the cells.

Although the trials are carried out with the anti-tumour antibody itselfor the substance produced by bonding a certain cytotoxic substance tothe anti-tumour antibody, the method has not been carried outpractically, because the anti-tumour antibody is a heteroprotein tohuman, since the antibody has been obtained by immunizing aheterospecific animal. Namely, in the case where the antibody obtainedfrom a heterospecific animal is administered to a person, the occurrenceof a serum disease such as anaphylaxis, etc. on the second andsuccessive administration cannot be evitable. In short, such ananti-tumour antibody can be used only once, and such a fact is thelargest demerit of the method. It is necessary to use a homospecificantibody for overcoming the demerit, and the monochronal antibodyprepared by using human lymphocytes is the ideal, however, such anantibody has not been provided.

Accordingly, it has been necessary for improving the demerits andsolving the affairs concerning the practicality of such a method toinvestigate the homospecific antibodies and select those assembling tothe tumour cells.

The present inventors, as a result of examination of the distribution ofthe various ¹²⁵ I-labelled antibodies in living body, have found thatthe general natural antibodies arrive at the portion of tumour andremain there for a long period of time and accordingly, the presentinventors have known that in the case where after bonding an anti-tumouragent to the immunoglobulin, the thus obtained substance is administeredto the cancer-bearing individual, the anti-tumour agent remains for along period in the portion of the tumour, thereby exhibiting theanti-tumour effect. The present invention has been attained on thesefindings.

The anti-tumour substance obtained by bonding an anti-tumour agent tohuman immunoglobulin according to the present invention has the largestspecificity and merit in that the anti-tumour substance obtained bybonding the anti-tumour agent to human immunoglobulin can be frequentlyadministered as compared to the known product produced by bonding theanti-tumour agent to the anti-tumour antibody derived from the animal ofdifferent species from human, and remains at the portion of tumour for along period of time.

Accordingly, the present invention provides a new type of medicines,which contains the anti-tumour substance produced by bonding ananti-tumour agent to human immunoglobulin and is highly practical.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided ananti-tumour substance obtained by bonding an anti-tumour agent to humanimmunoglobulin.

In a second aspect of the present invention, there is provided a processfor producing an anti-tumour substance comprising dissolving theanti-tumour agent in an aqueous solvent, after adding a bonding agent inthe thus formed aqueous solution, adding human immunoglobulin in thethus prepared aqueous solution, thereby reacting the globulin with theanti-tumour agent at -30° to 50° C. for one min to 48 hours, andpurifying the thus formed reaction product by at least one means ofpurification selected from the group consisting of salting out,precipitation, recrystallization, elution and column-fractionation, toproduce the anti-tumour substance obtained by bonding an anti-tumouragent to human immunoglobulin.

In a third aspect of the present invention, there is provided apharmaceutical composition in dosage unit form comprising as an activeingredient an anti-tumour substance obtained by bonding an anti-tumouragent to human immunoglobulin.

In a fourth aspect of the present invention, there is provided a methodfor treating tumour, which comprises administering an effective amountof a substance obtained by bonding an anti-tumour agent to humanimmunoglobulin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an anti-tumour substance obtained bybonding an anti-tumour agent to human immunoglobulin, and relates to aprocess for producing an anti-tumour substance comprising dissolving ananti-tumour agent in an aqueous solvent, after adding a bonding agent inthe thus prepared solution, adding human immunoglobulin into themixture, thereby reacting the human immunoglobulin with the anti-tumouragent at -30° to 50° C. for a period of from one min to 48 hours,subjecting the thus formed reaction product to at least one of thepurifying procedures selected from the group consisting of salting-out,precipitation, recrystallization, elution and column-fractionation,thereby obtaining the anti-tumour substance in which the anti-tumouragent has been bonded to human immunoglobulin, and further relates to apharmaceutical composition in dosage unit form comprising as an activeingredient the anti-tumour substance in which the anti-tumour agent hasbeen bonded to human immunoglobulin, and also relates to a method fortreating tumour, comprising administering an effective amount of theanti-tumour substance in which the anti-tumour agent has been bonded tohuman immunoglobulin.

The anti-tumour substance according to the present invention is theproduct of bonding a highly cytotoxic, anti-tumour agent belonging tothe group consisting of alkylating agents such as chlorambucil,melphalan, ACNU, cyclophosphamide and the like, antibiotics such asmitomycin C, doxorubicin hydrochloride, daunorubicin hydrochloride,bleomycin, actinomycin D, neocarcinostatin and the like, andantimetabolites such as cytarabine, 8-azaguanine, 5-fluorouracil,methotrexate, sodium aminopterin, 6-mercaptopurine and the like to humanimmunoglobulin under extremely mild reaction conditions, and is ananti-tumour substance which is excellent in anti-tumour effects in spiteof its remarkably low cytotoxicity as compared to that of theanti-tumour agent which is one of the starting materials of the presentsubstance.

Recently, various anti-tumour agents have been broadly used, therebyexhibiting a certain degree of effects. As the anti-tumour agent,chlorambucil, melphalan, ACNU, cyclophosphamide, cytarabine,8-azaguanine, 5-fluorouracil, methotrexate, sodium aminopterin,mitomycin, daunorubicin, actinomycin D and sarkomycin are used, however,each of these substances has a high cytotoxicity, and it has been knownthat after being administered with the substance, a side effect such asleucocytepenia, depilation and gastroenteric disorder appears on thepatient administered with the substance and accordingly, there isactually a limit of using the substance as a medicine.

On the other hand, trials have been carried out wherein an antibody tothe tumour cell or the tumour-related antigen is prepared or isolatedand the thus prepared or isolated cells or antigen is used in thetreatment of the tumour, however, favorable effects have not beenobtained. Furthermore, quite recently, it has been proposed that ananti-tumour effect is expected in a new substance obtained by chemicallybonding an anti-tumour agent to an anti-tumour antibody. However, sincethe conditions of reaction for obtaining such a substance are toosevere, any sufficient results have not been obtained. Further, in theabove-mentioned experiment, antibody obtained from an animal differentin species from human was used and accordingly, the side effect of theheterospecific antibody such as serum disease was inevitable.

Accordingly, the present inventors have found a method for purifying theanti-tumour antibody obtained from a heterospecific animal by affinitychromatography (refer to Japanese Patent Application Laying-Open Nos.55-092325 (1980), 56-065828 (1981) and 56-065829 (1981)). By the thusfound method, a highly purified antibody is obtained, however, there isstill a problem in the case of carrying out frequent administration.

As a result of examining the attainability of several antibodies to theportion of a tumour, it has been found by the present inventors that anaturally obtainable antibody obtained by affinity chromatography canattain the portion of the tumour at a high concentration and can remaintherein for a longer period of time in other organs, and based on theirfinding, each of the following anti-tumour agents was bonded to humanimmunoglobulin:

Chlorambucil, melphalan, ACNU, cyclophosphamide, cytarabin,8-azaguanine, 5-fluorouracil, methotrexate, sodium aminopterin,mitomycin C, doxorubicin hydrochloride, bleomycin, daunorubicin,actinomycin D and sarkomycin.

The thus obtained substances were found to exhibit respectively afavorable anti-tumour effect, and because of the easiness of synthesisand high stability of melphalan and esters thereof, the most preferablebonded substance was those derived from melphalan and esters thereof.For reference, the naturally obtainable antibody contains humanimmunoglobulin (Ig) and an antibody (F(ab')₂) of a low molecular weight.

The bonding of human immunoglobulin and an anti-tumour substance iscarried out by the following method.

The anti-tumour agent to be bonded to human immunoglobulin is dissolvedin an aqueous solvent selected from an aqueous acidic solution, anaqueous alkaline solution, an aqueous neutral solution and an aqueousphosphoric buffer solution containing sodium borate and the like. Abonding agent selected from the group consisting of carbodiimide,dextran, glutaraldehyde, diethylmalonimidate, isocyanate andpolyglutamic acid is added to the solution, and the reaction is carriedout by adding human immunoglobulin (containing also F(ab')₂) into themixture at -30° to +50° C., preferably 0° to 30° C. for one min to 48hours, preferably 10 min to 25 hours.

The reaction product was purified by a means of salting-out,precipitation, recrystallization, elution, column fractionation, therebyobtaining the anti-tumour substance comprising the anti-tumour agentbonded to human immunoglobulin.

The acute toxicity of the thus obtained anti-tumour substance(hereinafter referred to as the present substance) was examined byinjecting thereof intravenously to a group of mice at a dose rate of4000 mg/kg, however, no death could be observed in a week afterinjection.

The acute toxicity was further examined on the anti-tumour substancesobtained by bonding an anti-tumour agent to the antibody of lowmolecular weight which has been obtained by enzymatic hydrolysis ofhuman immunoglobulin with pepsin (refer to Nisonoff, Science, 132,1770(1970)), plasmin (Sgouris, Vox Sang, 18, 71(1967)), thermolysin(refer to Japanese Patent Application Laying Open No. 51-95125), papain,trypsin or chymotripsin. In the above-mentioned case of bonding theanti-tumour substance to the antibody of low molecular weight, forinstance, F(ab')₂, the intravenous injection of 4000 mg/kg thereof didnot kill the thus treated mice within a week.

Accordingly, the present substance is extremely low toxic and can beadministered frequently, and it is effective in treatment of humancancers, for instance, acute leukemia, malignant lymphoma, carcinoma,sarcoma, malignant chorionepthelioma, acute myelogenous leukemia,melanoma, acute lymphatic leukemia and myeloma; head and neck cancer,upper gastrointestinal cancer, colorectal carinoma, pancreatic carinomaand malignant hepatomas, endocrine tumors, genitourinary tumors,gynecological tumors, breast cancer, leukemias and myeloma and malignantmelanoma; and lung cancer, esophagus cancer, breast cancer, stomachcancer, colon cancer, rectal cancer, hepatic cancer (hepatoma), renalcancer, ovarian cancer, uterus cervical cancer and leukemias andmyeloma.

As a method of formulating in the case where the present substance isused as an anti-tumour agent or an active ingredient of a pharmaceuticalcomposition, and as a method of administering the composition, therespective known methods can be applied. As a route of administration,oral, injective or rectal may be mentioned. As the form of thecomposition comprising the present substance, powder, granule, tablet,injection and suppository may be mentioned, however, the administrationis preferably carried out by the tablet or injection. For formulatingthe composition for injection, an aqueous solvent such as aqueousphysiological saline solution, sterilized water, Ringer's solution,etc., non water-soluble solvents, isotonic agents, indolentic agent,stabilizers, antiseptics, suspension stabilizer, buffer, emulsifier maybe optionally used therein.

An example for formulation of the composition according to the presentinvention is shown as follows.

After preparing 50 ml of an aqueous solution of 1 g of the presentsubstance and 5 g of mannitol and sterilizing the solution in anordinary method, the sterilized solution is divided into vials forinjection, or freeze-dried as it is to be a composition for injection,which is diluted with an aqueous physiological saline solution oninjection. Generally, the content of the present substance in thecomposition is 0.01 to 90 % by weight, preferably 0.1 to 60% by weight.

The amount of administration of the present substance depends on theconditions of the patient, however, it is generally 0.1 to 10 g/day/60kg, preferably 1 to 6 g/day/60 kg.

According to the present invention, since the anti-tumour activity ofthe anti-tumour agent and the tumour-tropic property of the humanimmunoglobulin of the enzyme-treated human immunoglobulin are retainedin the anti-tumour substance of the present invention without losing,the present substance, when administered, arrives at the intended siteof tumour effectively, remains there for a long period of time toexhibit the anti-tumour effect.

Since it is not necessary to select the antibody from the anti-tumourantibody in the case of the synthesizing the present substance, thepresent invention is to be very advantageous industrially.

The present invention will be more precisely explained while referringto Examples as follows.

However, the present invention is not restricted to Examples undermentioned. From the foregoing description, one skilled in the art caneasily ascertain the essential characteristics of the present invention,and without departing from the spirit and scope thereof, can makevarious changes and modifications of the present invention to adapt itto various usages and conditions.

EXAMPLE 1 Examination of the Distribution of Human Immunoglobulin

In order to examine what kind of the antibodies is practically usableaccording to the process of the present invention, the distribution ofeach of the ¹²⁵ I-labelled-anti-S-180 rabbit immunoglobulin, ¹²⁵I-labelled-normal ICR mouse immunoglobulin and ¹²⁵ I-labelled-humanimmunoglobulin was administered to a S-180 cancer-bearing mouse, and thedistribution of each of the administered ¹²⁵ I-labelled immunoglobulinswas detected as follows.

Each of the immunoglobulins was labelled by ¹²⁵ I in the protein moietythereof by the method of W. H. Hurter et al. (refer of Biochem. J., 89,114 (1963)). For instance, mitomycin C bonded to anti-S-180 antibody(rabbit immunoglobulin) was dissolved in aqueous 0.5M phosphoric acidbuffer solution of pH of 7.4 so as to obtain a concentration of 5 mg/mland after introducing 0.5 ml of the solution in a Spick tube, Na¹²⁵ Iwas added to the solution in an amount of 0.25 mCi. To the mixture, 0.7mg of chloramin T dissolved in 200 μl of aqueous 0.05M phosphoric acidbuffer solution was added to carry out a reaction at 0° C. for 15 min.Thereafter, by adding 10 mg of potassium iodide and 1.75 mg of sodiumpyrosulfite dissolved in aqueous 0.05M phosphoric acid buffer solutionto stop the reaction. By passing the reaction mixture through a columnof Sephadex G-25 (2.2 cm in inner diameter and 40 cm in height), theun-reacted radioactive iodine and the reagents were removed to obtain¹²⁵ I-labelled mitomycin bonded to anti-S-180 rabbit immunoglobulin. Inthe same manner, ¹²⁵ I-labelled mitomycin bonded to normal ICR mouseimmunoglobulin and ¹²⁵ I-labelled mitomycin bonded to humanimmunoglobulin.

To a group of S-180 cancer-bearing ICR mice (after 2 weeks oftransplantation of S-180 cancer), each of the thus prepared ¹²⁵I-labelled mitomycin C bonded to the respective immunoglobulins wasintravenously administered, and after sacrificing the animals after 24and 144 hours of the administration, the blood, the portion of S-180cancer, the liver, the kidney, the intestine were extirpated and theradioactivity of the thus extirpated part or organ was examined byγ-counter of well-type to calculate the amount of each of the thusprepared ¹²⁵ I-labelled mitomycin C bonded to the respectiveimmunoglobulin per unit weight of the part or the organ.

From the thus obtained amounts, the distribution of the substance inliving body of the thus treated animal after 24 hours of administrationwas known as are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                    Ratio*.sup.2                                                       antibodyKind of                                                                      in tumour*.sup.1Accumulation                                                             ##STR1##                                                                                ##STR2##                                                                               ##STR3##                                ______________________________________                                        1*.sup.3                                                                             3.6        6.0       9.0      12.0                                     2      0.6        2.8       1.5      8.4                                      3      1.9        1.9       3.8      19.0                                     ______________________________________                                         Notes:                                                                        ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                 

In the same manner, by counting the radioactivity of the organsextirpated after 144 hours of administration, the percentage by weightof the remaining substance in each organ to the total amount ofremaining substance in the body of the animal was obtained as shown inTable 2.

                  TABLE 2                                                         ______________________________________                                        Unit: % by weight                                                             Organs or the site of the tumour                                              Kind of                                                                              Portion of                    Intes-                                   antibody                                                                             tumour    Liver   Kidney                                                                              Spleen                                                                              tine  Others                             ______________________________________                                         .sup. 1*.sup.1                                                                      66.6       7.4    11.1   5.6  1.9   7.4                                2      41.3      27.9     4.2  20.9  2.1   3.6                                3      38.7      10.2    20.4  10.2  2.0   18.5                               ______________________________________                                         Note: *.sup.1 :                                                               1: AntiS-180 rabbit immunoglobulin                                            2: Normal ICRmouse immunoglobulin                                             3: Human immunoglobulin.                                                 

The results show that the heterospecific antibody obtained by immunizingthe heterospecific animal with a tumour antigen exhibits an excellentattainability to the tumour, however, it is also shown that the naturalantibody of the same species remains in the portion of the tumour at ahigh rate as compared to the other organ, although the rate ofattainability of the natural antibody is as low as 1/5 to 1/10 ascompared to the attainability of the species-specific antibody. Fromthese facts, it has been known by the present inventors that the naturalantibody is high in practicality as a carrier.

EXAMPLE 2 Preparation of Human Immunoglobulin

In 1000 ml of serum of a normal healthy person, 1000 ml of aqueous0.005M phosphoric acid buffer solution of sodium chloride (hereinafterreferred to as PBS) were added to prepare a diluted serum, and to thediluted serum, 2000 ml of aqueous saturated solution of ammonium sulfatewas added slowly under agitation. After leaving the mixture to standstill for 60 min at 4° C., a precipitate was salted out, and bysubjecting the mixture to centrifugal separation at 8,000 rpm for 30min, the precipitate was collected and dissolved in PBS to make thewhole volume to 1,000 ml. By adding 250 ml of aqueous saturated solutionof ammonium sulfate to the mixture, the degree of saturation of ammoniumsulfate of the whole mixture was made to 20%. In the case where themixture became turbid to form a precipitate, the whole mixture wassubjected to centrifugation to remove the precipitate which wasfibrinogen. Into the thus obtained supernatant liquid, 250 ml of aqueoussaturated solution of ammonium sulfate were added to make the degree ofsaturation by ammonium sulfate of the whole mixture to 33%. Afterleaving the whole mixture to stand still for 60 min, the whole mixturewas subjected to centrifugation at 8,000 rpm for 30 min to collect theprecipitate. After dissolving the thus collected precipitate in 1,000 mlof PBS, 500 ml of aqueous saturated solution of ammonium sulfate wereadded to the solution. After stirring the whole mixture for 60 min, thewhole mixture was subjected to centrifugation at 8,000 rpm for 30 min tocollect the precipitate, and the thus collected precipitate wasdissolbed in 300 ml of PBS. The solution was dialyzed against PBS toremove ammonium sulfate, and the dialyzate was subjected to columnchromatography while using DEAE-cellulose column of 5 cm in diameter and50 cm in height, thereby obtaining a fraction passed through at 0.005Mand pH of 8.0. The fraction passed through the column was dialyzedagainst distilled water to remove the salt contained therein, and thedialyzate was subjected to freeze-drying to obtain 12.5 g of humanimmunoglobulin.

EXAMPLE 3 Preparation of the Substance Obtained by BondingImmunoglobulin Derived from Normal Human to an Anti-Tumour Agent

Each of mitomycin C, doxorubicin hydrochloride, daunorubicilhydrochloride, breomycin, actinomycin and sarkomycin was reacted withimmunoglobulin derived from normal human to synthesize each of thepresent substances as shown in the following.

Bonding of Mitomycin C to Human Immunoglobulin

In 100 ml of distilled water, 1.0 g of human immunoglobulin wasdissolved, and in the thus formed solution, 111.3 mg of mitomycin C wasdissolved. After adjusting the pH of the solution to 5.5 with aqueous1.0N hydrochloric acid, 262.6 mg of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was addedthereto at 4° C. and after the following reaction time, the reaction wasstopped by the addition of 5 ml of an acetic acid-sodium acetate buffersolution of pH of 5.5. After condensing and desalting the reactionmixture while using an ultrafilter to 10 ml, 10 ml of the thus obtainedcondensate was passed through a column of Sephadex G-25 (made byPharmacia Japan Co.) of 5 cm in diameter and 90 cm in height tocompletely separate the high molecular weight substance and the lowmolecular weight substance. The supernatant solution obtained bysubjecting the eluate to ultracentrifugation for 60 min at 40,000 G wasfreeze-dried at 0° C. to obtain the present substance as a product. Theamount of bonded mitomycin C was determined by ultraviolet absorption at360 nm and shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Reaction time                                                                 (hour)    Mitomycin C/human immunoglobulin (μg/mg)                         ______________________________________                                        1         2.8                                                                 4         5.3                                                                 24        8.6                                                                 ______________________________________                                    

In the same manner as above, 1.0 g of human immunoglobulin was broughtinto reaction with each of doxorubicin hydrochloride, daunorubicinhydrochloride, bleomycin and actinomycin D to obtain about 800 mg of therespective substances.

The amount of bonded doxorubicin per mg of human immunoglobulin was 4.8μg (in the case of reacting for 60 min) and 9.5 μg (in the case ofreacting for 24 hours).

EXAMPLE 4

Each of the present substances was prepared by reacting immunoglobulinderived from a normal human with each of chlorambucil, melphalan(phenylaranine mustard), ACNU, uramustin, cyclophosphamide and melphalanmethyl ester, according to amide-bonding as is shown in the following.

(4-1) Bonding Melpharan to Human Immunoglobulin

In 100 ml of distilled water, 1.0 g of human immunoglobulin wasdissolved, and 100 mg of melphalan were suspended in the thus formedsolution. After adjusting the pH of the aqueous suspension to 5.5 withthe addition of aqueous 1.0N hydrochloric acid,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was addedto the aqueous suspension to react for 24 hours. After stopping thereaction with the addition of 5 ml of aqueous acetic acid-sodium acetatebuffer solution, the reaction mixture was condensed and desalted whileusing an ultrafilter to a volume of 10 ml and the condensate (10 ml) waspassed through a column of Sephadex G-25 (made by Pharmacia Japan Co.)of 5 cm in diameter and 90 cm in height, thereby completely separate thehigh-molecular weight substances and the low-molecular weightsubstances.

After subjecting the eluate to ultracentrifuge of 40,000 G for 60 min,the supernatant liquid was freeze-dried at 0° C. to obtain the presentsubstance. The content of protein in the thus obtained present substancewas determined by the Copper-Folin Method while using albumin as thestandard, and the alkylation activity of the present substance wasdetermined by the method of Epstein (refer to Epstein, J. Anal. Chem.,27, 1423 (1955)), and as the result, it was found that 6 μg of melpharanbonded to 1 mg of human immunoglobulin.

(4-2) Bonding Chlorambucil, ACNU or Uramustine to Human Immunoglobulin

In the similar procedures to that in 4-1, 1.0 g of human immunoglobulinwas brought into reaction to each of chlorambucil, ACNU and uramustineto obtain about 900 mg of each of the present substances. The amount ofchlorambucil bonded to 1 mg of human immunoglobulin was 5.1 μg afterreacting for 60 min and 11.7 μg after reacting for 24 hours.

(4-3) Bonding Methyl Ester of Melphalan to Human Immunoglobulin

After dissolving 1.0 g of human immunoglobulin in 100 ml of distilledwater, 100 mg of hydrochloride of methyl ester of melphalan weredissolved in the thus prepared solution, and while adjusting the pH ofthe solution to 5.5 with the addition of aqueous 1.0N hydrochloric acidsolution, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloridewas added to react for 24 hours. After stopping the reaction with theaddition of 5 ml of aqueous acetic acid-sodium acetate buffer solutionof pH of 5.5 to the reaction mixture, the reaction mixture was condensedand desalted to 10 ml by the use of an ultrafilter. Ten milliliters ofthe thus condensed and desalted liquid was passed through a column ofSephadex G-25 (made by Pharmacia Co.) of 5 cm in diameter and 90 cm inheight, thereby separating the high-molecular weight substances and thelow-molecular weight substances in the reaction liquid completely. Theeluate was subjected to ultracentrifugation at 40,000 G for 60 min, andthe supernatant liquid was freeze-dried at 0° C. to obtain the presentsubstance. The amount of methyl ester of melpharan bonded to 1 mg ofhuman immunoglobulin was 10 μg.

EXAMPLE 5

Immunoglobulin derived from normal human was reacted with each ofcytarabine, 8-azaguanine, 5-fluorouracil, methotrexate and aminopterinsodium to obtain each of the compounds forming an amide bonding as shownin the following example.

(5-1) Bonding of Methotrexate to Human Immunoglobulin

After dissolving 1.0 g of human immunoglobulin in 100 ml of distilledwater, 151.3 mg of methotrexate were dissolved in the thus preparedsolution, and while adjusting the pH of the solution to 5.5 with theaddition of aqueous 1.0N hydrochloric acid solution,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was addedto react for 24 hours. After stopping the reaction with the addition of5 ml of aqueous acetic acid-sodium acetate buffer solution of pH of 5.5to the reaction mixture, the reaction mixture was condensed and desaltedto 10 ml by the use of an ultrafilter. Ten milliliters of the thuscondensed and desalted liquid was passed through a column of SephadexG-25 (made by Pharmacia Co.) of 5 cm in diameter and 90 cm in height,thereby separating the high-molecular weight substances and thelow-molecular weight substances in the reaction liquid completely. Theeluate was subjected to ultracentrifugation at 40,000 G for 60 min, andthe supernatant liquid was freeze-dried at 0° C. to obtain the presentsubstance. The amount of methotrexate bonded to 1 mg of humanimmunoglobulin determined by utilizing the ultraviolet absorption at 305nm was 10 μg.

(5-2) Bonding of Cytarabine, 8-Azaguanine, 5-Fluorouracil or AminopterinSodium to Human Immunoglobulin

After dissolving 1.0 g of human immunoglobulin in 100 ml of distilledwater, 100 mg of each of cytarabine, 8-azaguanine, 5-fluorouracil andaminopterin sodium were dissolved in the thus prepared solution, andwhile adjusting the pH of the solution to 5.5 with the addition ofaqueous 1.0N hydrochloric acid solution,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was addedto react for 24 hours. After stopping the reaction with the addition of5 ml of aqueous acetic acid-sodium acetate buffer solution of pH of 5.5to the reaction mixture, the reaction mixture was condensed and desaltedto 10 ml by the use of an ultrafilter. Ten milliliters of the thuscondensed and desalted liquid was passed through a column of SephadexG-25 (made by Pharmacia Co.) of 5 cm in diameter and 90 cm in height,thereby separating the high-molecular weight substances and thelow-molecular weight substances in the reaction liquid completely. Theeluate was subjected to ultracentrifugation at 40,000 G for 60 min, andthe supernatant liquid was freeze-dried at 0° C. to obtain about 900 mlof each of the present substances. The amount of cytarabine bonded to 1mg of human immunoglobulin was 4.7 μg after reaction of 60 min and 8.3μg after reaction of 24 hours.

EXAMPLE 6 Bonding of an Anti-tumour Agent to Human ImmunoglobulinF(ab')₂ (6-1) Preparation of Human Immunoglobulin F(ab')₂

After dissolving 1 g of human immunoglobulin into 100 ml of aqueous 0.1Nsodium acetate buffer solution of pH of 4.5, pepsin was added to thethus prepared solution so as to make the weight ratio of the enzyme tothe protein to be 1:100 in the solution, and the digestion was carriedout at 37° C. for 16 hours. After stopping the reaction by adding solidtris-hydrochloride so as to make the pH of the reaction mixture to 8.0,the reaction liquid was condensed to 10 ml by the use of an ultrafilter.Five milliliters of the condensate were charged into a column ofSephadex G-150 (made by Pharmacia Co.) of 5 cm in diameter and 90 cm inheight and eluted the adsorbed matter by PBS of pH of 7 of the thusobtained three peaks, the first peak was collected as F(ab')₂. Afterfilling the thus collected fraction into a dialysis tube, the fractionwas subjected to desalting and then freeze-drying to obtain humanimmunoglobulin F(ab')₂.

(6-2) Bonding an Anti-tumour Agent to Human Immunoglobulin F(ab')₂

After dissolving 500 mg of the thus obtained human immunoglobulinF(ab')₂ in 50 ml of distilled water, 55.6 mg of mitomycin C weredissolved in the thus prepared solution. While adjusting the pH of thesolution to 5.5 with the addition of aqueous 1.0N hydrochloric acidsolution, 131.3 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride was added to the solution at 4° C. to make the reagentsreact together for the under-mentioned time period, and then thereaction was stopped by the addition of 5 ml of aqueous aceticacid-sodium acetate buffer solution. After condensing the reactionliquid to 5 ml by the use of an ultrafilter, the condensate was passedthrough a column of Sephadex G-25 (made by Pharmacia Co.) of 5 cm indiameter and 90 cm in height to completely separate the high-molecularweight substances and the low-molecular weight substances in thecondensate. After subjecting the eluate to ultracentrifugation at 40,000G for 60 min, the supernatant liquid was freeze-dried at 0° C. to obtainthe present substance. The amount of mitomycin C bonded to humanimmunoglobulin F(ab')₂ determined by utilizing the ultravioletabsorption at 360 nm was shown in Table 4-1 as a function of thereaction time.

                  TABLE 4-1                                                       ______________________________________                                        Reaction time                                                                           Mitomycin C/Human immunoglobulin F(ab').sub.2                       (hour)    (μg/mg)                                                          ______________________________________                                        1         3.4                                                                 4         6.7                                                                 24        10.3                                                                ______________________________________                                    

(6-3) Bonding Another Anit-tumour Agent to Human Immunoglobulin F(ab')₂

In a quite similar manner to that in 6-2, each of the anti-tumour agentsshown in Table 4-2 was reacted with human immunoglobulin F(ab')₂ toobtain each of the present substance.

                  TABLE 4-2                                                       ______________________________________                                        Anti-tumour agent                                                                              Yield of the present substance                               ______________________________________                                        Doxorubicin hydrochloride                                                                      about 800 mg                                                 Daunorubicin hydrochloride                                                                     about 800 mg                                                 Bleomycin        about 800 mg                                                 Actinomycin D    about 800 mg                                                 Chlorambucil     about 400 mg                                                 Melphalan        about 400 mg                                                 ACNU             about 400 mg                                                 Uramustine       about 400 mg                                                 Methyl ester of Melphalan                                                                      about 400 mg                                                 Cyclophosphamide about 400 mg                                                 Cytarabine       about 400 mg                                                 8-Azaguanine     about 400 mg                                                 5-Fluorouracil   about 400 mg                                                 Methotrexate     about 400 mg                                                 Aminopterin sodium                                                                             about 400 mg                                                 ______________________________________                                    

In addition, the respective amounts of doxorubicin hydrochloride,melphalan and methotrexate to one milligram of humna immunoglobulinF(ab')₂ as the respective functions of reaction time were shown in Table4-3.

                  TABLE 4-3                                                       ______________________________________                                        Unit: μg                                                                                   Reaction time (hour)                                          Anti-tumour agent 1        24                                                 ______________________________________                                        Doxorubicin hydrochloride                                                                       8.5      19.6                                               Melphalan         8.1      17.6                                               Methotrexate      7.5      17.3                                               ______________________________________                                    

EXAMPLE 7 Production of the Present Substance by the Use of Dextran andGlutaraldehyde as the Bonding Agent

In 500 ml of distilled water, 500 mg of dextran was dissolved, and afteradjusting the pH of the solution to 11 by the addition of aqueous 1Nsodium hydroxide solution, a solution of 250 mg of bromcyan in 1 ml ofacetonitrile was rapidly added to the alkaline solution under agitationand the pH of the mixture was adjusted to 10.8 to 11.0 by the additionof aqueous 1N sodium hydroxide solution and the pH was maintained for 10min after addition of bromcyan. Thereafter, 100 mg ofhexamethylenediamine dissolved in 2.5 ml of water were added to themixture and the pH of the whole mixture was adjusted to 9.0 by theaddition of aqueous 1N hydrochloric acid solution.

After stirring the mixture for 5 min, 250 mg of melphalan were added tothe mixture, and the pH of the reaction mixture was reduced to 6.5 bythe addition of aqueous 1N hydrochloric acid and the reduced pH wasmaintained for 15 min. Then, the reaction mixture was condensed at 4° C.to 10 ml. The whole amount of the condensate was passed through a columnof Sephadex G-25 (made by Pharmacia Japan Co.) of 5 cm in diameter and90 cm in height to separate the substance of high molecular weight andthe substance of low molecular weight completely, and the eluate wassubjected to ultracentrifugation at 40,000 G for 60 min and the thusobtained supernatant liquid was freeze-dried at 0° C. to obtain acompound formed by bonding 30 to 50 molecules of melphalan to onemolecule of dextran.

By using 100 mg of the thus obtained compound and 100 mg of humanimmunoglobulin F(ab')₂ while bonding the two by glutaraldehyde, one ofthe present substance according to the present invention was obtained.

In a quite similar manner to that shown above except for using 250 mg ofmitomycin C instead of 250 mg of melphalan, a compound formed by bonding30 to 50 molecules of mitomycin C to one molecule of dextran wasobtained, and using 100 mg of the thus obtained compound and 100 mg ofhuman immunoglobulin F(ab')₂, another of the present substance wasobtained in the presence of 100 g/ml of glutar aldehyde in the system atroom temperature.

In a quite similar manner to that shown above except for using 250 mg ofmethotrexate instead of 250 mg of mitomycin C, a compound formed byadding 30 to 50 molecules of methotrexate to one molecule of dextran wasobtained. By bonding 100 mg of the thus obtained compound to 120 mg ofhuman immunoglobulin F(ab')₂ in the presence of 100 μg/ml ofglutaraldehyde (at the final concentration), still another one of thepresent substances according to the present invention was obtained. Inaddition, by using human immunoglobulin instead of human immunoglobulinF(ab')₂, a different one of the present substances was obtained.

EXAMPLE 8 Production of the Present Substance by Only Using GlutarAldehyde as the Bonding Agent

In 1 ml of aqueous 0.01M phosphoric acid buffer solution of pH of 6.8,11.3 mg of mitomycin C were dissolved, and 20 μl of an aqueous 1% byweight solution of glutaraldehyde was added to the thus preparedsolution, and the mixture was stirred for 8 hours at room temperature.Then, a solution of 100 mg of human immunoglobulin dissolved in 20 ml ofaqueous phosphoric acid buffer solution of pH of 6.8 was added to themixture to carry out the reaction for 2 hours at room temperature. Thereaction mixture was passed through a column of Sephadex G-25 (made byPharmacia Japan Co.) of 5 cm in diameter and 90 cm in height to separatethe substance of high molecular weight and the substance of lowmolecular weight completely. After subjecting the eluate toultracentrifugation at 40,000 G for 60 min, the supernatant liquid wasfreeze-dried at 0° C. to obtain one of the present substances in which9.6 μg of mitomycin C was bonded to 1 mg of human immunoglobulin.

In a quite similar manner to that shown above except for using 196 mg ofadriamycin instead of 11.3 mg of mitomycin C, another one of the presentsubstances was obtained in which 5.6 μg of adriamycin was bonded to onemg of human immunoglobulin.

In a quite similar manner to that shown above except for using 200 mg ofmethotrexate instead of 11.3 mg of mitomycin C, and using 100 mg ofhuman immunoglobulin F(ab')₂ instead of 100 mg of human immunoglobulin,still another one of the present substances was obtained in which 7.8 μgof methotrexate was bonded to one mg of human immunoglobulin F(ab')₂.

EXAMPLE 9 Production of the Present Substance by Diethylmalonimidate asa Bonding Agent

In 10 ml of aqueous 0.2M solution of sodium borate of pH of 9.3, 11.3 mgof mitomycin C and 100 mg of human immunoglobulin were dissolved.Thereto 5 mg of diethylmalonimidate was added, and the mixture wasstirred for one hour at room temperature while keeping the pH of themixture at 8.6. Then, 2.5 mg of diethylmalonimidate was further added tothe reaction mixture and the stirring was carried out for one hour.After the reaction was over, the reaction mixture was brought to neutraland by adding aqueous saturated solution of ammonium sulfate to thereaction mixture, a compound obtained by bonding human immunoglobulin tomitomycin C was precipitated. After collecting the precipitate bysubjecting the reaction mixture to centrifugation at 7,000 rpm, theprecipitate was dissolved in 5 ml of aqueous 5 mM phosphoric acid buffersolution, and the thus obtained solution was dialyzed against distilledwater until no ammonium sulfate became detected in the dialyzate (for 72hours). The dialyzate was passed through a column of Sephadex G-25 ) of5 cm in diameter and 90 cm in height to remove the substance of lowmolecular weight in the dialyzate completely. The eluate wasfreeze-dried at -20° C. to obtain one of the present substances of thepresent invention, in which 6.3 g of mitomycin C bonded to one mg ofhuman immunoglobulin.

In a quite similar manner to that shown above, another one of thepresent substances according to the present invention wherein mitomycinwas bonded to human immunoglobulin F(ab')₂ was obtained.

In a quite similar manner to that shown above, each of the presentsubstances was obtained from 100 mg of human immunoglobulin and one ofmelphalan and aminopterin sodium in a yield of 85 mg.

EXAMPLE 10 Examination of Anti-Tumour Activity of the Present Substance

Examination of the anti-tumour activities of the present substancesobtained by bonding an anti-tumour agent to human immunoglobulin orobtained by bonding the anti-tumour agent to human immunoglobulinF(ab')₂ was carried out by using three kinds of tumours while comparingto the anti-tumour activity of the anti-tumour agent as follows.

(10-1) Anti-tumour Activity to Sarcoma 180 Solid Tumour

Cells of mouse sarcoma-180 which had been subjected to repeatedsubculture on ICR mouse were transplanted subcutaneously in axillarypart of each of 10 ICR mice of a group at a rate of 1×10⁶ cells/animal,and to each of the thus treated mice, an aqueous solution of each of theknown anti-tumour agents, an aqueous solution of each of the presentsubstances respectively produced by bonding one of the known anti-tumouragents to human immunoglobulin or an aqueous solution of each of thepresent substances respectively produced by bonding one of the knownanti-tumour agents to human immunoglobulin F(ab')₂ wasintraperitoneously administered every other day for 10 times in total.After 5 days of the final administration, the mouse was sacrificed toextirpate the tumour(s) to be weighed. The average weight of thetumours(T) of the thus treated mice and the average weight of thetumours(C) of the control mice administered with an aqueousphysiological saline solution instead of the agent or the presentsubstance were treated in the following formula, thereby obtaining theanti-tumour activity of the administered substance, i.e., the activityof suppressing the proliferation of the transplanted tumour: ##EQU1##

The thus obtained anti-tumour activity of the present substance producedin Examples 3 to 6 is shown in Table 5 while comparing that of theanti-tumour agent used for producing the present substance.

                                      TABLE 5                                     __________________________________________________________________________    Anti-tumour Activity (Activity of suppressing                                 the proliferation of Sarcoma-180 Solid Tumour)                                                   Amount of administration                                                      (mg/kg b.w.) Anti-tumour                                                          Calculated as the                                                                      activity                                      Substance administered                                                                           Total                                                                             anti-tumour agent                                                                      (%)                                           __________________________________________________________________________    Mitomycin          1   1        40                                            Mitomycin + human immunoglobulin                                                                 200 1        35                                            Mitomycin + human immunoglobulin                                                                 200 1        38                                            F(ab').sub.2                                                                  Bleomycin          0.5 0.5      45                                            Bleomycin + human immunoglobulin                                                                 100 0.5      40                                            Bleomycin + human immunoglobulin                                                                 100 0.5      42                                            F(ab').sub.2                                                                  Doxorubicin        2   2        50                                            Doxorubicin + human immunoglobulin                                                               400 2        47                                            Doxorubicin + human immunoglobulin                                                               400 2        49                                            F(ab').sub.2                                                                  __________________________________________________________________________

(10-2) Anti-tumour Activity to Yoshida Sarcoma

Ascites cells of Yoshida sarcoma which had been subjected to subculturerepeatedly while using Donryu rats were transplanted into the abdominalcavity of each of 10 Donryu rats of a group at the rate of 1×10⁶cells/animal, and from after 24 hours of the transplantation, an aqueoussolution of an anti-tumour agent, an aqueous solution of a substanceobtained by bonding the anti-tumour agent to human immunoglobulin or anaqueous solution of a substance obtained by bonding the anti-tumouragent to human immunoglobulin F(ab')₂ was administered every other dayfor 5 times in total intraperitoneously to the thus transplanted rat.Rats were observed to record the average survival days(T)(for ratsadministered with each agent), and the average survival days(C)(for ratsadministered only with an aqueous physiological saline solution insteadof the agent). The rate of life-elongation was calculated by theformula: ##EQU2##

The results are shown in Table 6.

                                      TABLE 6                                     __________________________________________________________________________    Anti-tumour Activity (Rate of Life-Elongation)                                                    Amount of administration                                                      (mg/kg b.w.)  Rate of                                                              Calculated as the                                                                      Life-Elongation                             Substance administered                                                                            Total                                                                              anti-tumour agent                                                                      (%)                                         __________________________________________________________________________    Chlorambucil        1.0  1.0      259                                         Chlorambucil + human immunoglobulin                                                               200  1        230                                         Chlorambucil + human immunoglobulin*                                                              200  1        235                                         Melphalan           1.5  1.5      230                                         Melphalan + human immunoglobulin                                                                  300  1.5      200                                         Melphalan + human immunoglobulin*                                                                 300  1.5      190                                         Melphalan methyl ester + human                                                                    300  1.5      220                                         immunoglobulin                                                                Melphalan methyl ester + human                                                                    300  1.5      230                                         immunoglobulin*                                                               Uramustine          5    5        290                                         Uramustine + human immunoglobulin                                                                 1000 5        240                                         Uramustine + human immunoglobulin*                                                                1000 5        260                                         __________________________________________________________________________     Note:                                                                         *means human immunoglobulin F(ab').sub.2                                 

(10-3) Anti-tumour Activity to Mouse Leukemia P-388

Ascites cells of mouse leukemia P-388 which had been subjected torepeated subculture while using DBA/2 mouse were intraperitoneouslytransplanted to each of 10 DBA/2 mice of a group, and from after 24hours of the transplantation, an aqueous solution of each of theanti-tumour agent, an aqueous solution of each of the substance obtainedby bonding each of the anti-tumour agent to human immunoglobulin or anaqueous solution of each of the substance obtained by bonding each ofthe anti-tumour substance to human immunoglobulin F(ab')₂ wasintraperitoneously administered once a day for continuous 5 days. Thethus treated mice were observed to record the average survival days(T)of the mice administered with the agent or the substance, and theaverage survival days(C) of the control mice (administered with anaqueous physiological saline solution instead of the agent or thesubstance). The anti-tumour activity of the agent or the substance (Rateof Life-elongation) was calculated by the following formula. ##EQU3##

The results are shown in Table 7.

                                      TABLE 7                                     __________________________________________________________________________    Anti-tumour Activity (Rate of Life-Elongation)                                                    Amount of administration                                                      (mg/kg b.w.)  Rate of                                                              Calculated as the                                                                      Life-Elongation                             Substance administered                                                                            Total                                                                              anti-tumour agent                                                                      (%)                                         __________________________________________________________________________    Cytarabine          20   20       220                                         Cytarabine + human immunoglobulin                                                                 4000 20       190                                         Cytarabine + human immunoglobulin*                                                                4000 20       195                                         Methotrexate        3    3        250                                         Methotrexate + human immunoglobulin                                                               600  3        220                                         Methotrexate + human immunoglobulin*                                                              600  3        235                                         Aminopterin sodium  3    3        270                                         Aminopterin sodium + human immuno-                                                                600  3        240                                         globulin                                                                      Aminopterin sodium + human immuno-                                                                600  3        250                                         globulin*                                                                     Melphalan           1.5  1.5      230                                         Melphalan + Dextran + Human                                                                       15   1.5      210                                         immunoglobulin                                                                Melphalan + Dextran + Human                                                                       15   1.5      220                                         immunoglobulin*                                                               Adriamycin          2    2        50                                          Adriamycin + Human immunoglobulin                                                                 20   2        48                                          Adriamycin + Human immunoglobulin*                                                                20   2        46                                          Mitomycin C         1    1        40                                          Mitomycin C + Human immunoglobulin                                                                10   1        35                                          Mitomycin C + Human immunoglobulin*                                                               10   1        38                                          __________________________________________________________________________     Note:                                                                         *Human immunoglobulin F(ab').sub.2                                       

What is claimed is:
 1. An anti-tumor substance manufactured by a processcomprising:(a) dissolving melphalan or ester thereof in aqueous solvent;(b) adding a bonding agent, selected from the group consisting ofcarbodiimides, glutaraldehyde, diethylmalonimidate, isocyanate andpolyglutamic acid, to the aqueous solution, and then adding humanimmunoglobulin obtained from a normal healthy person to said aqueoussolution, and thereby reacting said human immunoglobulin and saidmelphalan or ester thereof at a temperature of -30° to 50° C. for 1minute to 48 hours; and (c) subjecting the thus produced reactionproduct to purification by means of a method selected from the groupconsisting of salting out, precipitation, recrystallization, elution andcolumn-fractionation.
 2. The anti-tumor substance according to claim 1,wherein said bonding agent is a carbodiimide.
 3. The anti-tumorsubstance according to claim 2, wherein said carbodiimide is oneselected from the group consisting of 1-ethyl-3,3-dimethylaminopropylcarbodiimide, dicyclohexyl carbodiimide and morpholinoethylcarbodiimide.
 4. The anti-tumor substance according to claim 1, whereinsaid ester is a lower alkyl ester.
 5. The anti-tumor substance accordingto claim 4, wherein said lower alkyl ester is a methyl ester.
 6. Theanti-tumor substance according to claim 1, wherein said humanimmunoglobulin is IgG.
 7. The anti-tumor substance according to claim 1,wherein said human immunoglobulin is F(ab')₂.
 8. A pharmaceuticalcomposition in dosage unit form comprising an effective amount of ananti-tumor substance obtained by bonding melphalan or ester thereof tohuman immunoglobulin derived from a normal healthy person using abonding agent, selected from the group consisting of carbodiimides,glutaraldehyde, diethylmalonimidate, isocyanate and polyglutamic acid,as the active ingredient thereof.
 9. The pharmaceutical compositionaccording to claim 8, wherein said bonding agent is carbodiimide. 10.The pharmaceutical composition according to claim 9, wherein saidcarbodiimide is one selected from the group consisting of1-ethyl-3,3-dimethylaminopropyl carbodiimide, dicyclohexyl carbodiimideand morpholinoethyl carbodiimide.
 11. The pharmaceutical compositionaccording to claim 8, wherein said ester is a lower alkyl ester.
 12. Thepharmaceutical composition according to claim 11, wherein said loweralkyl ester is a methyl ester.
 13. The pharmaceutical compositionaccording to claim 8, wherein said human immunoglobulin is IgG.
 14. Thepharmaceutical composition according to claim 8, wherein said humanimmunoglobulin is F(ab')₂.
 15. A method for treating a tumor selectedfrom the group consisting of human cancers of breast, lung, esophagus,stomach, colon, kidney, prostate, gallbladder and bone marrow, whichcomprises administering an effective amount of a compound obtained bybonding melphalan or ester thereof to human immunoglobulin derived froma normal healthy person using a bonding agent selected from the groupconsisting of carbodiimides, glutaraldehyde, diethylmalonimidate,isocyanate and polyglutamic acid.
 16. The method according to claim 15,wherein said bonding agent is a carbodiimide.
 17. The method accordingto claim 16, wherein said carbodiimide is one selected from the groupconsisting of 1-ethyl-3,3-dimethylaminopropyl carbodiimide, dicyclohexylcarbodiimide and morpholinoethyl carbodiimide.
 18. The method accordingto claim 15, wherein said ester is a lower alkyl ester.
 19. The methodaccording to claim 18, wherein said lower alkyl ester is a methyl ester.20. The method according to claim 15, wherein said human immunoglobulinis Ig G.
 21. The method according to claim 15, wherein said humanimmunoglobulin is F(ab')₂.